The present invention relates to novel compositions of matter and methods for the treatment of respiratory distress with these novel compositions. This invention also relates to the chemical modification of the polypeptides or protein fragments which enhance the surfactant-like properties of phospholipids. More specifically, the present invention relates to the covalent attachment of fatty acids of various chain lengths to polypeptides comprising fragment replicas and analogs of fragment replicas of the naturally occurring low molecular weight hydrophobic surfactant associated proteins known as SP-B and SP-C and to their use in the formulation of novel medicaments useful in the establishment, modification and/or maintenance of pulmonary surface tension.
Specifically incorporated herein, by reference for purposes of establishing the background of the present invention, are the teachings and disclosures of the following U.S. Patent Applications:
1. U.S. patent application Ser. No. 860,239, filed May 6, 1986; PA0 2. U.S. patent application Ser. No. 060,719, filed Jun. 10, 1987; PA0 3. U.S. patent application Ser. No. 101,680, filed Oct. 1, 1987; PA0 4. U.S. patent application Ser. No. 397,151 filed Aug. 22, 1989.
Also incorporated herein by reference are U.S. Pat. No. 4,659,805 and U.S. Pat. No. 4,882,422 which disclose and claim a high molecular weight surfactant protein known as SP-A. Also incorporated herein by reference is U.S. Pat. No. 4,918,161 which discloses and claims a low molecular weight surfactant protein known as SP-B.
In general, these references disclose the discovery, method of isolation, characterization and use of a family of naturally occurring mammalian surfactant-associated proteins. Members of this family have been designated as SP-A, SP-B and SP-C. These proteins are known to have the capacity to effect the surfactant-like activity of both natural and synthetic phospholipids. It should be noted that the associated scientific literature also uses the nomenclature of SAP-B, SAP-(Phe), SAP-6 (Phe), and SPL-(Phe) for SP-B. SP-C is also referred to as SAP-C, SAP-(Val), SAP-6 (Val) and SPL (Val) in the prior art. These two proteins (SP-B and SP-C) are distinct gene products with unique amino acid sequences. Both proteins are derived from proteolytic processing of larger precursor proteins synthesized by pulmonary type II epithelial cells.
SP-B is generated by cleavage of the precursor protein at a glutamine-phenylalanine peptide bond resulting in the naturally occurring protein having 78 amino acid residues, with an N-terminal residue of phenylalanine and a simple molecular weight of about 8,700. SP-B isolated from human lung migrates on polyacrylamide gels as an entity having a relative molecular weight (M.sub.r) of 7-8,000 after sulfhydryl reduction. Without sulfhydryl reduction the naturally occurring protein is found as large oligiomers. SP-B is extremely hydrophobic, a physical property which is consistent with its in vivo strong association with phospholipids and solubility in organic solvents such as chloroform and methanol.
SP-C has an amino terminal glycine or phenylalanine residue, a molecular weight of about 3,700, a polyvaline sequence, and, like SP-B, is also extremely hydrophobic. In addition, both proteins (SP-B and SP-C) are substantially resistant to enzyme degradation by proteases such as trypsin, chymotrypsin, staphylococcus nuclease V-8, endoglycosidase F, and collegenase. Neither SP-B nor SP-C exhibits any degradation or alteration in their molecular weight distribution following treatment with these enzymes. In this behavior, as well as on the basis of amino acid sequence information, the proteins are clearly different from the more hydrophilic and higher molecular weight protein SP-A (also known as SAP-35).
SP-A is present in natural lung surfactant material and has a reduced molecular weight of 30-36,000. SP-A is a glycoprotein containing an internal collagen-like region which is rich in glycine and hydroxyproline. This protein has a N-linked complex carbohydrate and a calcium binding site in the C-terminal globular domain. SP-A is known to bind to phospholipids and is thought to confer important structural organization to the surfactant lipids. This protein is also believed to play a role in preventing the inhibition of pulmonary surfactant activity by plasma or other proteins.
The complete amino acid sequence of SP-B and SP-C has been determined from amino acid analysis and deduced from DNA's derived from the mRNA's encoding the proteins. The SP-B and SP-C proteins are available as isolates from natural sources, such as bronchioalveolar lung washes and minced lung tissue or as products resulting from the application of recombinant DNA methodologies. When formulated with phospholipids (including synthetic phospholipids) these proteins provide compositions useful in the treatment of pulmonary disorders.
As is often the case with biologically active substances, the isolation of substantial quantities of hydrophobic SP-B and SP-C proteins from natural sources is expensive and labor intensive. Production of these proteins by recombinant DNA techniques requires substantial effort in terms of design and achieving optimal host/vector expression systems to facilitate production of the proteins. In addition, considerable effort is required to develop effective isolation strategies to separate and purify the expressed protein of interest from the unwanted material. Solid phase peptide synthesis is a feasible alternative for obtaining both SP-B and SP-C. However, in either production scenario the low molecular weight, extreme hydrophobicity and large number of valine residues markedly complicates commercial exploitation of the material.
The principal difficulty arises from the extreme hydrophobicity and hence markedly limited solubility of the SP-C polypeptide (regardless of its mode of production) resulting from its primary amino acid sequence and high valine content. As a consequence it is necessary to utilize chemically and clinically unacceptable solvents, (e.g. concentrated formic acid) to solubilize the polypeptide. Complete removal of such solvents is not only necessary in order to minimize oxidative damage to other components of the admixture, but is also difficult owing to the low vapor pressure of these solvents. As a consequence extremely time consuming and laborious strategies need to be developed.
The medical community has a need for commercial quantities of SP-C which can be readily utilized in pharmaceutical formulations (i.e. admixtures with phospholipids and the like). The present invention fulfills that need through the discovery that covalent attachment of various fatty acids (FA) to SP-C markedly improves the solubility of the FA-SP-C conjugates in solvents such as ethanol and/or methanol.
The usefulness of the naturally occurring SP-B and SP-C proteins resides in their ability to significantly improve the surface tension lowering capacity and respreadability of phospholipid admixtures. Natural SP-B and SP-C have been shown, both individually as well as in combination, to facilitate this improvement in surfactant-like activity of phospholipids. However, use of the unmodified SP-C sequence (i.e. no covalently attached fatty acid) results in admixtures preparations having variable surface properties. It is believed this is due to batch-to-batch variation in SP-C solubility.
The prior art fails to suggest, disclose or contemplate the instant discovery which is, in part, the conjugation of a fatty acid to SP-C. Further one skilled in the art can not a priori predict which fatty acids when covalently attached to SP-C will evidence utility or that certain FA-SP-C conjugates will have activity exceeding that of the complete unmodified natural protein or polypeptide.
It is thus clear that FA-SP-C conjugates or analogs thereof, would provide numerous advantages over the use of unmodified SP-C produced by chemical or recombinant synthesis. These advantages include ease and reproducibility of admixture formulation and significant cost savings resulting from the ability to utilize polar solvents. The FA-SP-C conjugates can be combined with phospholipids or with natural SP-B, or with recombinantly expressed SP-B, solid phase synthetic full length SP-B, or fragments and/or analogs thereof to produce useful formulations.